| Background and objectiveEsophageal cancer(EC)is the eighth most common cancer with a high mortality of the sixth most leading cause of cancer related death worldwide.According to the histopathology feathers,EC is mainly divided into esophageal adenocarcinoma(EA)and esophageal squamous cell carcinoma(ESCC).ESCC remains predominant EC especially in China.Although surgery is the main treatment of early-stage esophageal cancer,radiotherapy is still the predominant treatment for the patients with late-stage EC(especially ESCC)or with no tolerance or willing of surgery.Radiotherapy has many advantages in the ESCC treatment including local tumor control.However,radioresistace always happens and becomes a challenging obstacle for ESCC treatment.So it is meaningful to make out the molecular mechanisms of radioresistance and find possible strategies for increasing cellular radiosensitivity.Nowadays,radiotherapy in combination with targeted therapy has become a focus of radiosensitization.In phase 3 clinical trials,radiation in combination with the EGFR inhibitor cetuximab has been proved to achieve a survival benefit of 10%in HNSCC;In preclinical models,PI3K/mTOR inhibitor has been demonstrated to enhance HNSCC radiosensitivity.Apart from these two signaling pathways that promote radioresistance,deregulation of Wnt signaling is found to be associated with radioresistance of multiple cancers.Here we generated two radioresistant cells rECA109 and rKyse150 from parental esophageal cancer cells ECA109 and Kyse150.We then found that Wnt signaling activity was higher in radioresistant cells and inhibiting Wnt signaling could reverse the radioresistance of rECA109 and rKyse150 cells.In our present study,high-mobility group box 1 protein(HMGB1),a chromatin associated protein,was firstly found to be transactivated by β-catenin/TCF4 complex and promote chromatin remodel and the activation of DNA damage checkpoint response after IR treatment,thereby providing "access",and "time" for the DNA damage repair machine.Our research aims to uncover the tight correlation between Wnt signaling and radioresistance and provide possible target for increasing ESCC radiosensitivity.Materials and Methods1.Two radioresistant cells rECA109 and rKyse150 were generated by fraction irradiation for a total of 60Gy.To determine the radioresistance levels,clonogenic survival assays were performed and survival curves were then drawn.D0 and Dq values were calculated using single-hit multi-target model.In addition,animal experiment was performed to further demonstrate the generation of radioresistant cells;2.Identification of EMT in radioresistant cells:cell morphology change was observed under microscope;WB analyzed EMT representative protein expression(E-cadherin,Vimentin and Slug);Transwell assays assessed the migration potential.Identification of cancer stem cell-related quality:CD133 expression by Flow cytometry and tumor sphere formation assay;3.To analyze Wnt activity,WB and IF staining were performed to assess P-catenin contents and localization;4.To determine the correlation between Wnt signaling and radioresistance,after iCRT14 treatment for radioresistant cells and WNT1 treatment for parental cells,clonogenic survival assays were then performed;5.To compare DNA damage repair capacity between parental and radioresistant cells,IF staining was performed to assess p-yH2AX foci levels Oh,0.5h and 24h after 1R treatment;6.To determine the correlation between Wnt signaling and DNA damage repair,24h after IR treatment,p-yH2AX foci levels were assessed in parental cells treated by WNT1 or sterile water and radioresistant cells treated by iCRT14 or DMSO via IF staining.In addition,0.5h after IR treatment,acetyl-H3 and DNA damage checkpoint protein(p-Chk1,p-cdc25C and ATRIP)were assessed in parental cells treated by WNT1 or sterile water and radioresistant cells treated by iCRT14 or DMSO via WB;7.qRT-PCR analyzed iCRT14 or WNT1-induced chromatin modifier genes mRNA expression change;8.ChIP demonstrated that β-catenin/TCF4 complex bound with HMGB1 promoter,thereby transactivating HMGB1;9.Vectors expressing HMGB1 or HMGB1 shRNA were constructed to transfect parental cells and radioresistant cells respectively.Clonogenic survival assays were performed to assess the correlation between HMGB1 and radioresistance.p-yH2AX foci were assessed by IF staining in ECA109/NC,ECA109/HMGB1,rECA109/SiNC and rECA109/SiHMGB1cells 24h after IR treatment;acetyl-H3 levels were assessed by WB in ECA109/NC,ECA109/HMGB1,rECA109/SiNC and rECA109/SiHMGBlcells 0.5h after IR treatment;10.Rescue experiment:after iCRT14 treatment for rKyse150/HMGB1 and rKyse150/NC cells and WNTltreatment for rKyse150/SiHMGBland rKyse150/SiNC cells,Clonogenic survival assays were performed to demonstrate HMGB 1 mediated Wnt-induced radioresistance;IF staining(p-yH2AX foci)was performed to HMGB1mediated Wnt-induced DNA damage repair;WB(p-Chkl,p-cdc25C and ATRIP)was performed to HMGBlmediated Wnt-induced the activation of DNA damage checkpoint response.Results1.Two radioresistant cells rECA109 and rKysel50 were generated by fraction irradiation for a total of 60Gy.DO and Dq values of radioresistant cells were significantly higher than parental cells(P<0.01).Animal experiment further demonstrated the generation of redioresistant cells;2.Radioresistant cells acquired EMT properties:Spindle morphology,up-regulation of Vimentin and Slug,down-regulation of E-cadherin and enhanced migration capacity.In addition,radioresistant cells acquired cancer stem cells associated properties:up-regulation of CD 133 and sphere formation;3.In the absence of IR,β-catenin mainly distributed the membrane of ECA109 and Kyse 150.However,P-catenin partly entered the nucleus of rECA109 and rKyse 150.After IR exposure,the nuclear β-catenin was significantly increased in radioresistant cells while there were just little changes of the nuclear β-catenin in parental cells;4.iCRT14 increased radiosensitivity of radioresistant cells,while WNT1 promoted radioresistance of parental cells;5.DNA damage repair capacity of radioresistant cells was up-regulated;6.iCRT14 inhibited DNA damage repair of radioresistant cells,while WNT1 promoted DNA damage repair of parental cells after IR treatment;7.iCRT14 inhibited chromatin remodel and the activation of DNA damage checkpoint response in radioresistant cells,while WNT1 promoted chromatin remodel and the activation of DNA damage checkpoint response in parental cells after IR treatment;8.β-catenin/TCF4 complex were bound with the promoter of HMGB1,thereby transactivating HMGB1;9.HMGB 1 promoted radioresistance;10.HMGB 1 promoted DNA damage repair with the activation of DNA damage checkpoint response after IR treatment;11.HMGBlpartly mediated Wnt-induced radioresistance and DNA damage repair.Conclusion1.Wnt signaling promoted histone modification and the activation of DNA damage checkpoint response after IR treatment,thereby enhancing DNA damage repair and inducing radioresistance thereafter;2.β-catenin/TCF4 complex were bound with the promoter of HMGB1,thereby transactivating HMGB 1;3.HMGB1partly mediated Wnt-induced radioresistance and DNA damage repair. |
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